Research Advances Role of STIM2 in SOCE and Regulation of Cell Function: This year we have directed considerable efforts to efforts to resolve the exact role of STIM2 in regulating SOCE and cell function. We have examined how STIM2 facilitates (i) STIM1 clustering and (ii) STIM1-gating of Orai1 at relatively high ER-Ca2+, i.e. conditions that likely do not elicit a STIM1 response. There is considerable evidence to show that conformational changes in STIM1 C-terminus are critical for STIM1/Orai1 coupling. Such information is not available for STIM2. 1. STIM2 Induces Activated Conformation of STIM1 to Control Orai1 Function in ER-PM Junctions. - Ca2+ entry mediated by the calcium channel, Orai1, provides critical Ca2+ signals that regulate cell function. The ER-Ca2+ sensor protein, STIM1, recruits and strongly activates Orai1 within ER-PM junctions. STIM2 is a poor activator of Orai1, and its physiological role is not well understood. Herein, we report a crucial function for STIM2 in inducing the activated conformation of STIM1. By using conformational sensors of STIM2 and STIM1, together with protein interaction and functional studies, we show that STIM2 is constitutively localized within ER-PM junctions in ER-Ca2+ store replete cells. We showed that STIM2 adopts an open conformation even in unstimulated cell. Importantly, STIM2 traps STIM1 and triggers remodeling of STIM1 C terminus, causing STIM1/Orai1 coupling and enhancement of Orai1 function in cells with relatively high ER-Ca2+. The increase in Ca2+ entry controls Ca2+-dependent transcription factor, NFAT, activation at low agonist. Our findings reveal that STIM2 modulates STIM1/Orai1 function to tune the fidelity of receptor-evoked Ca2+ signaling and the physiological response of cells. 2. STIM2 compartmentalizes Orai1+STIM1 in functionally competent ER-PM junctions where Orai1 activity is coupled with NFAT activation. - ER-PM junctions where STIM/Orai assembly occurs also determine the coupling of Ca2+ entry to downstream effectors. We have examined the role of STIM2 and STIM1-polybasic domain in targeting channel assembly in these functionally competent domains. Depletion of ER-Ca2+ store induces assembly of Orai1+STIM1 complex in ER-PM junctions resulting in Ca2+ entry and enhanced nuclear translocation of NFAT. STIM1K, which lacks the polybasic C-terminal domain and does not cluster on its own, is also recruited by Orai1 to ER-PM junctions following ER-Ca2+ depletion and induces Ca2+ entry that is similar to that with Orai1+STIM1. However, NFAT activation is minimally enhanced. Expression of STIM2 with either Orai1+STIM1 or Orai1+STIM1K results in clustering of all three proteins in ER-PM junctions. Importantly, expression of STIM2 with Orai1+STIM1K rescues NFAT activation to levels seen with Orai1+STIM1, without affecting Ca2+ entry. Knockdown of STIM2 or STIMATE (TMEM110, required for clustering of STIM1) decreases both SOCE and NFAT activation. Unlike STIM2, expression of STIMATE with Orai1+STIM1K does not induce recovery of NFAT activation. Interestingly, expression of STIM2 in siSTIMATE-treated cells rescues the clustering of Orai1+STIM1, SOCE and NFAT activation. These findings suggest that Orai1+STIM1K assemble in ER-PM junctions that do not permit coupling of Ca2+ entry to NFAT activation. STIM2 recruits Orai1+STIM1K into ER-PM junctions where channel function is coupled with regulation of NFAT. Together, our findings demonstrate that STIM2 recruits and compartmentalizes Orai1/STIM1 in functionally competent ER-PM junctions which permit coupling of Orai1 function with NFAT activation. This is being further studied to see whether STIM2 promotes association of Orai1 with AKAP79 and calcineurin, key proteins that mediate coupling of Orai1 function with NFAT activation. STIM2 proteins modulate the dynamic range of Orai1/STIM1 function - The recently identified STIM2 displays poor interaction with Orai1 and is reported to attenuate SOCE. Its Ca2+ sensitivity is similar to that of STIM2 and it also clusters with STIM1 and STIM2. Its SOAR domain has an additional sequence that is proposed to interfere with activation of Orai1. Our previous studies revealed that STIM2 can recruit Orai1 and STIM1 into ER-PM junctions and trigger a conformational change in STIM1. This results in STIM1-mediated Orai1 activation in cells with high ER-Ca2+. In this study, we have examined the effect of STIM2 on STIM1/Orai1 function. Our findings show that while STIM2 does not activate Orai1, it promotes STIM1-gating of Orai1. STIM2 when co-expressed withSTIM1, but not with STIM1-L394H or STIM1-R426L, caused constitutive activation of endogenous Orai1. Activation of endogenous Orai1, was increased in the following order: STIM2 < STIM2+STIM2 STIM2 < STIM2+STIM1 < STIM2+STIM1. STIM1 activates Orai1 only after store depletion. We suggest that the Orai1-mediated Ca2+ entry with STIM2+STIM1 is lower than that with STIM2+STIM1 due to the contribution of a relatively weaker gating of Orai1 by STIM2. Importantly, our findings show that although STIM2 and STIM2 exert differential effects on Orai1, both proteins recruit and trigger STIM1/Orai1 coupling. Current experiments are designed to assess the role of STIM2 in cell function; agonist-stimulated Ca2+ signals, NFAT activation, in vivo in salivary gland fluid secretion (ad-viruses under preparation), and assessment of dimeric vs heteromeric interactions of STIMs. Assessment of dynamic status of endogenous STIM/Orai proteins. - The major components of SOCE are Orai1, STIM1, and STIM2. Upon ER-Ca2+ store depletion, the STIM proteins undergo a conformational change and translocate to distinct sites (ER-PM junctions) at the cell periphery, where they interact with the Ca2+ channel Orai1 that is localized in the plasma membrane and activate Ca2+ entry. These dynamic changes in the STIM proteins have been studied in great detail. However, till date all studies reporting clustering patterns of the proteins within ER-PM junctions, have been done using overexpression systems utilizing tagged proteins. Most of these use strong promoter based expression systems. Thus the status of endogenous proteins and how they respond to store-depletion is not known. For example, the temporal characteristics of their mobilization, the size/nature of endogenous STIM clusters within ER-PM junctions for all three components of SOCE have not yet been studied. We have utilized CRISPR/Cas9 technique to knock-in a fluorescent tag (mVenus) into the endogenous STIM2-N terminus in HEK293 and HEK293T cells. Initial studies show that this protein appears to form constitutive dynamic clusters, which increase when cells are stimulated. These findings are consistent with our previous findings, although the size and the mobility of the clusters vary. We are now using plasmids with relatively weak-promoter as a basis for comparison. Studies are now progressing in the laboratory to examine the activation dynamics of the endogenous STIM2 protein and its interaction with Orai1 and STIM1, and super-resolution imaging.